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Boos appear several times throughout the story in Paper Mario. most Boos inhabit Boo's Mansion, but several yellow Boos live in Gusty Gulch. All the Boos are led by Lady Bow, who ends up joining Mario on his journey in Chapter 3. In Boo Mansion, several Boos test Mario at the request of Lady Bow. A Boo can be seen guarding a chest with a weight in it, which must be used to progress through the Mansion. Another Boo has a record, which must be used to make the Boo guarding the chest flee. A circle of Boos is also found in the basement guarding the Super Boots.

In Gusty Gulch, the Boos that live there are being eaten by Tubba Blubba. Mario heads off to try a stop Tubba Blubba, and several of the Boos assist him.

In The Thousand-Year Door, Boos inhabit the Creepy Steeple. When Mario first enters the Steeple, only one lonely Boo is found, who forces Mario to open a chest with 200 Boos in it. He will then ask Mario how many boos were in the chest and, if Mario answers correctly, the Boo will give him an Ultra Shroom. If Mario answers incorrectly, he is given a Coin. If Mario talks to the Boo again, he will ask if Mario is going to be mean to him. If the response is no, then the Boo will summon a swarm of other Boos that kicks him out of the Mansion. If the answer is yes, then Mario can enter an optional boss fight with the Atomic Boo.

Boos return in World 4 of Paper Mario: Sticker Star. They roam around The Enigmansion. When Mario is in the Mansion, he must find all the Boos and seal them in the Book of Sealing. Several different types of Boos are found during Mario's search, such as a 10-Stack and a crumpled Boo. There is also a Golden Boo, which drops a significant amount of coins when defeated. Once all of them have been sealed, a Big Boo is fought.

In Color Splash, Boos are commonly found in the Purple Region. They inhabit the parallel universes, and are commonly found in stacks of 10. One Boo can also be seen in the hot spring found in Redpepper Crater, but he does not harm Mario.